Prop questions: Why is a new blank needed for this exercise? 2. Calculate the nu
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Prop questions: Why is a new blank needed for this exercise? 2. Calculate the nunber of HnoI in (sample calculation: Lab 1, P5) oa 33 mk solution. | 3. To determine the kinetic effects of the enzyme reaction, you will need to determine the time required to produce 10 mol dopachrome in the 3 mL volume within the cuvette. In other words, the end point of your reaction will be represented by the concentration of dopachrome reaching a value of 10 umol/3 mL or 3.3 mM (3.3 umol/mL) The absorbance for a concentration (c) of 3.3 m can be calculated by the Beer-ambert law (A-ec), using the extinction coefficient (e) determined in exercise 5.2 Therefore, based an your calculated extinction coefficient, an absorbance of dopachrome from DOPA in you 3 mL reaction mixture. will be required to convert 10 ol Exercise 5.3: Kinetic Analysis of Phenoloxidase 1. Prepare a 1/10 dilution of your enzyme extract by placing 0.5 mL extract into 4.5 mL O.1 M citrate buffer 2. Label this tube and place the 1/10 dilution in the ice bath 3. Prepare a blank using 0.5 mL of your enzyme diluteion and 2.5 5. Add 0.5 mL of the appropriately diluted enzyme extract and pH 6.6 until ready. mL 0.1 M citrate buffer pH 6.6 and reblank the spec. 4. Add 2.5 mL of 10 mM DOPA to a clean Spec tube immediately record the time. Mix and immediately insert the tube into the Spectrophotometer 6. Record the absorbance values at 0.5 min intervals and record them in Table 5.3. Continue readings until 8 min is reached. ATA ANALYSIS: in mol (y-axis) in the 3 mL reaction mixture. (Sample calculation; Lab 1-P5) (include in manual) 1. Plot time in min (x-axis) versus the amount of dopachrome 2. Using this plot determine the time required for the min production of 10 pmol of dopachrome.Explanation / Answer
It's important to know the principle behind the preparation of blanks.
Blanks are the spectrophotometer's equivalent of the tare function that is present on the measuring scale. If you are weighing a substance in a beaker, you cancel out the weight of the beaker and you simply consider the remaining value i.e. weight of the substance. The same goes with blanks.
There is a specific substance's absorbance that is sought in the experiment. Each chemical you add to the reacting test tube will absorb some light of its own. You don't want this value to screw up your results and hence you will make a blank which contains all the chemical except the substance whose absorbance is desired.
In the first experiment the substance whose absorbance was desired was phenoloxidase. Hence it goes by the above principle that it should not be added in the blank. hence the blank consists of citrate buffer.
In order to understand why a second blank is needed in the second experiment, notice the subtle difference between both the experiments. the second experiment is measuring something other than the absorbance of phenoloxidase. It is measuring the absorbance of the end product of a reaction at various time intervals. Since phenoloxidase is no more the desired substance, according to the principle of blanks, it must go in the blank cuvette. That is why you need to make a blank once again.
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